Activating mutations in KRAS occur frequently in some of the most common and deadliest of human cancers but efforts to target KRAS for therapeutic purposes have not been successful. KRAS generates two highly homologous isoforms (KRAS4A and KRAS4B) as a result of alternative splicing. While the complete Kras gene knockout in mice causes embryonic lethality, knock-in of either Kras4A (Kras4AKI) or Kras4B (Kras4BKI) at the expense of the alternate isoform results in viable mice. Kras4AKI mice express only Kras4A and Kras4BKI mice express only Kras4B, enabling for the first time a comparison of the individual roles of these Kras isoforms in transformation in vivo. We have found that Kras4BKI mice are highly resistant to the development Kras mutant lung and skin tumors, suggesting that Kras4A is essential for transformation in 2 independent tissues. Our studies further showed that Kras4A is also responsible for the previously reported tumor suppressor activity of wild type Kras. The overall goal of this proposal is to exploit these novel mouse models to study the functions of both Kras4A and Kras4B in lung cancer development, both as oncogenes (mutant form) and as tumor suppressors (wild type form). A major strength of this proposal is the availability of viable strains uniquely expressing each isoform, representing powerful and complementary in vivo systems for studies into the isoform specific functions of this major human oncogene. Aim 1 of this proposal will investigate the oncogenic function of individual Kras isoforms in vivo using models of carcinogen induced lung carcinogenesis. Aim 2 will investigate the tumor suppressor activity of wild type Kras4A and Kras4B in vivo in models of conditionally activatable mutant Kras (i.e. KrasLA2 and KrasLSL-G12D). Aim 3 will take advantage of lung epithelial cell lines derived from the various mouse models for detailed in vitro analyses of Kras4A and Kras4B signaling pathways in order to gain mechanistic insights into their oncogenic activities. Aim 4 will evaluate the roles of these individual KRAS isoforms in the growth/maintenance of human KRAS mutant lung cancer cells. Insights into the isoform specific functions of KRAS will not only be important to the understanding of cancer development but could potentially have major implications for therapeutic design.